Signal transduction through specific receptors, including integrins, mediates the coordinated cycles of cell attachment and release from the substratum which characterize migration. A2058 human melanoma cells migrate to gradients of soluble and insoluble vitronectin (Vn) through integrin alphavbeta3, the major Vn receptor on these cells. Migration is transduced through a G protein in the case of soluble Vn, and requires interaction with the RGD cell adhesion site in both soluble and insoluble Vn. Our goals have been to further elucidate the intracellular signaling pathways mediating motility to ECM proteins. Since ligation of integrins is known to induce tyrosine phosphorylation, we investigated the patterns of tyrosine phosphorylation in A2058 cells stimulated by soluble and insoluble Vn. When cells adhered to substratum-bound Vn, a limited number of proteins was phosphorylated on tyrosine, including a diffuse band of - 68 kDa. This band was subsequently identified as paxillin, a recently described focal adhesion protein. Its phosphorylation is alphavbeta3- dependent, and increases over time with degree of cell spreading, reaching a maximum at 45 minutes (-5X above starting level). Similarly, cells migrating over insoluble Vn demonstrated a continuous increase in paxillin tyrosine phosphorylation. Antibody to alphavbeta3, which inhibits migration, also reduces paxillin phosphorylation to the same extent (-50%). In contrast, soluble Vn at 50-100 microg/ml does not stimulate additional tyrosine phosphorylation of paxillin relative to background levels. Tyrosine phosphorylation of paxillin may therefore play a role in the cytoskeletal shape changes required for spreading and haptotactic migration to Vn. Cells migrating to soluble Vn, however, apparently modulate their shape in a distinct manner; therefore, signaling pathways for chemotaxis and haptotaxis may converge at a point downstream of paxillin tyrosine phosphorylation.